Reactive oxygen species (ROS) are generated inevitably in cells of all aerobic organisms, from bacteria plants and animals. Coping with the potentially damaging consequences of ROS generation is a problem of broad biological significance. The involvement of excited and highly reactive intermediates in oxygenic photosynthesis poses unique problems for algae and plants with respect to the generation of ROS. The proposed research plan is focused on the question of how photosynthetic organisms prevent or minimize photo-oxidative damage caused by ROS. To dissect antioxidant defenses in the chloroplast, mutants of the unicellular green alga Chlamydomonas reinhardtii, have been isolated. Several mutants affect the metabolism of xanthophyll pigments, which have critical roles in photoprotection in all algae and plants. An npq1 lor1 double mutant, which is deficient in lutein and zeaxanthin, undergoes photo-oxidative bleaching in high light, presumably due to increased production and/or decreased quenching of ROS, especially singlet oxygen. The proposed research will examine the types of photo- oxidative damage and the responses of various antioxidant systems in npq1 lor1 cells. Isolation of the genes affected in the xanthophyll mutants will enable determination of the molecular basis for the mutant phenotypes and will provide a strong foundation for further genetic analysis, including isolation of suppressors of npq1 lor1. The generation and/or scavenging of other ROS is perturbed in another group of mutants, which will be characterized using a combination of molecular genetic, biochemical, and physiological approaches. Understanding the metabolism and function of xanthophylls and other antioxidants in algae and plants will provide insights into the important roles of these molecules in preventing age-related diseases such as macular degeneration in humans and will enable engineering of antioxidant metabolism in plants to benefit human nutrition and health.